Nearest-neighbor parameters for the prediction of RNA duplex stability in diverse in vitro and cellular-like crowding conditions

Abstract

RNA performs various spatiotemporal functions in living cells. As the solution environments significantly affect the stability of RNA duplexes, a stability prediction of the RNA duplexes in diverse crowded conditions is required to understand and modulate gene expression in heterogeneously crowded intracellular conditions. Herein, we determined the nearest-neighbor (NN) parameters for RNA duplex formation when subjected to crowding conditions with an ionic concentration relevant to that found in cells. Determination of the individual contributions of excluded volume effect and water activity to each of the NN parameters in crowded environments enabled prediction of the thermodynamic parameters and their melting temperatures for plenty of tested RNA duplex formation in vitro and in cell with significant accuracy. The parameters reported herein will help predicting RNA duplex stability in different crowded environments, which will lead to an improved understanding of the stability-function relationship for RNAs in various cellular organelles with different molecular environments.

In-Cell Stability Prediction of RNA/DNA Hybrid Duplexes for Designing Oligonucleotides Aimed at Therapeutics

“…18 We recently demonstrated that 40 wt % PEG 200 could mimic the molecular environment of cellular organelles. 30 Hence, the thermodynamic parameters of the designed hybrid duplexes were measured under typical cellular conditions in the S1). Experimentally observed ΔG°3 7 and T m values were compared in the presence and absence of the cosolute (Table S1), showing a reduction of thermal stability from dilute to crowded conditions, as observed for DNA duplexes.…”

“…Thus, we propose eq 7 to determine ΔG°3 7NN,[crowder] by adding the individual contributions to NN parameters by the major factors from the excluded volume effect (ΔG°3 7NN,ev ) and water activity (ΔG°3 7NN,wa ) of the cosolute by following our previous study. 30…”

“…The thermodynamic parameters, such as the change in enthalpy, entropy, and free energy (Δ H °, Δ S °, and Δ G ° 37 , respectively) for hybrid duplex formation under molecular crowding conditions and the melting temperatures ( T m s), were determined using UV-melting studies as described previously . We recently demonstrated that 40 wt % PEG 200 could mimic the molecular environment of cellular organelles . Hence, the thermodynamic parameters of the designed hybrid duplexes were measured under typical cellular conditions in the presence of 40 wt % PEG 200 (Table S1).…”

“…As ΔG°3 7,wa was plotted against the changes in water activity, Δa w (Δa w = a w no cosolute − a w cosolute ), in Figure S14, the plot shows two linear correlations depending on the nature of the cosolutes, as observed for RNA duplexes. 30 Although disruption of the water network by adding cosolutes may destabilize the duplex, cosolutes containing hydroxyl groups in close vicinities, such as EG, Gly, and 1,3-PDO may form Hbonds with water molecules to help maintain the ordered water structure around the duplex, which reduces destabilization under crowding conditions. Based on the linear relations (Figure S14), ΔG°3 7NN,wa [crowder] and the corresponding prefactor m cs of the cosolutes were derived (Experimental Section in the Supporting Information), where m cs depends on the nature of the cosolutes and the NN base pairs (Table 2).…”

“…49 Gradual increase and decrease of I 520 and I 570 , respectively, with increasing temperature in the solutions with 0 to 40 wt % of PEG 200 were studied (Figure S16), as PEG 200 at various concentrations can mimic cellular conditions. 30 The calculated FE values were plotted against the temperature for intermolecular duplex formations (RDcontrol, RD1, RD2a, RD2b, and RD3) under diverse crowding conditions (Figure S17). The FE ratios (ratio of the FE value for the duplex to that of RDcontrol from Table S12) at 37 °C were assessed (Figure S18A) to neglect the effect of environmental conditions on the fluorescence intensity because RDcontrol did not dissociate at 37 °C for all of the conditions (Figure S17A).…”